Carotenoids, the basic source of yellow, orange and red, are among the most common, naturally occurring pigments. Carotenoids are naturally biosynthesized by bacteria, algae, fungi, and plants. Carotenoids have numerous benefits for human health and commercial utility as food flavorings, colorants, nutrient supplements, cosmetics and animal feed additives and supplements. The global market for carotenoids is growing, from $1.2 billion in 2010 to an estimated $1.4 billion by 2018 with CAGR of 2.3%. For example, β-carotene is used extensively in dietary supplements and as a colorant in food, beverage, and pharmaceutical formulations, and accounted for $261 million in 2010 and will be worth $334 million in 2018; lycopene is used for food color and food additive, dietary supplements, and accounted for $66 million in 2010 and will be worth $84 million in 2018. Carotenoids are produced commercially by chemical synthesis, extraction from natural sources, or microbial fermentation. Currently, over 90% of the market is covered by chemically synthesized carotenoids.
Products of carotenoid degradation such as α-ionone and β-ionone also have commercial importance. For instance, α-ionone and β-ionone are important fragrance chemicals that are used extensively in the perfumes and fragrance industry. In particular, α-ionone has a variety of applications, ranging from flavor and fragrance to cosmetics and pharmaceutical industries. α-ionone is an unsaturated ketone with a pleasant floral scent, and is naturally found in a variety of oils of flowers of Boronia Megastigma (Nees), renna, in violet moss and in oil of costus root, black currants, blackberries, raspberries, black tea, plum and peach. It is commonly used as a flavor agent, including in non-alcoholic beverages, ice cream, candy, gelatins and puddings, chewing gum, and as a fragrance agent in decorative cosmetics, and in nearly all perfumes.
There are three basic methods for obtaining α-ionone: 1) chemical synthesis; 2) direct extraction from a natural source; or 3) de novo biotechnological transformation, which includes microbial and enzymatic biotransformation. None of the currently available methods of producing α-ionone are satisfactory. Chemical synthesis currently dominates the global market. However, as α-ionone is a chiral compound, chemical synthesis produces a racemic mixture, (R)(+)-α-ionone and (S)(−)-α-ionone, which have different sensorial properties, and which are too costly to separate. In nature, α-ionone is found as an almost optically pure (R)(+)-enantiomer (>99%). As such, the sensorial properties of chemically produced α-ionone are not equivalent to the natural (R)(+)-α-ionone enantiomer. Additionally, chemical synthesis is environmentally unfriendly and is not accepted by consumers who like natural products.
Direct extraction from a natural source is also not very feasible. In general, the biological systems that naturally produce carotenoids are industrially intractable and/or produce the compounds at such low levels that commercial scale isolation is not practicable. For instance, plants have been an important source of natural ionones, but they carry ionones in such low amounts that the extraction is tedious and costly. The content of α-ionone is extremely low in plants, about 1.3-81 μg/kg in raspberry and blackberry. Additionally, there are no known natural biological systems capable of producing α-ionone alone. All known natural biological systems that produce ionones either produce β-ionone or a mixture of α- and β-ionone.
De novo synthesis of α-ionone is an attractive alternative for the production of α-ionone because it yields only the desirable enantiomer, is less damaging to the environment, and does not generate toxic waste. Most importantly, α-ionone produced by this method is defined as “natural” and demands a high market value. However, currently known de novo systems use enzymes from potentially harmful bacteria. Two exogenous genes (crtB and crtI) from Pantoea ananatis were used for lycopene production in Yarrowia lipolytica. Pantoea ananatis is an unconventional plant pathogen bacterium implicated in diseases of a wide range of host crops, including maize and onion, Eucalyptus, sudangrass and honeydew melons. Its implication in human infections reveals its capacity for proliferation and potential to cause disease in a vertebrate host. As such, the bacterium carries potential risks for humans and the environment, and enzymes from such bacteria that are to be used in food or medical applications are not accorded GRAS status (generally regarded as safe) for use in food or medical applications.
Therefore, there is a need for improved biological systems capable of efficiently providing natural, non-synthetic alternatives for carotenoids, and in particular the α-ionone carotenoid derivative, at a lower cost.